Development of hematopoiesis proceeds through two distinct steps, i.e. primitive and definitive hematopoiesis. In mice, primitive hematopoiesis begins in the extraembryonic yolk sac at 7.5 days post coitum (dpc) in gestation, while definitive hematopoiesis, which is distinguished by enucleated erythrocytes, lymphopoiesis, and generation of long term repopulating hematopoietic stem cells (LTR-HSCs), originates from the intraembryonic aorta-gonad-mesonephros (AGM) region at 10.5 to 11.5 dpc (Muller, A. M. et al. (1994) Immunity, 1, 291-301) (also reviewed by (Dzierzak, E. et al. (1998) Immunol. Today 19, 228-236; Keller, G et al. (1999) Exp. Hematol. 27, 777-787). Within 1 to 3 days of their emergence, LTR-HSCs migrate from the AGM region to the fetal liver and then emigrate to the bone marrow just before birth. Lymphopoietic cells and multi-potential hematopoietic progenitors are also detected in the para-aortic splanchnopleura (P-Sp) region of mouse embryos at 7.5 to 9.5 dpc (Cumano, A. et al. (1996) Cell 86, 907-916; Delassus, S., and Cumano, A. (1996) Immunity 4, 97-106; Godin, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 773-777), an intraembryonic site preceding the AGM region. However, LTR-HSCs, which are capable of repopulating lethally irradiated adult mice, have not been found in the P-Sp region. Interestingly, it was recently reported that LTR-HSCs can be detected in the yolk sac and the P-Sp region after transplantation into the livers of busulfan-treated newborn mice (Yoder, M. C. et al. (1997) Immunity 7, 335-344). Therefore, one speculation has been that LTR-HSCs generated in these sites lack homing capacity to the bone marrow and that the phenotypic differences in hematopoiesis between the yolk sac, the P-Sp region, and the AGM region can be mostly attributed to the supporting microenvironment. However, it still remains unknown how LTR-HSCs in the yolk sac acquire full repopulation activity.
Early in the last century, detailed observations of the early development of chick embryos led to the hypothesis that hematopoietic cells and endothelial cells arise from a common precursor termed the hemangioblast (Murray, P. D. F. (1932) Proc. Roy. Soc. London 11, 497-521; Sabin, F. R. (1920) Contributions to Embryology 9, 213-262) [also reviewed by (Wagner, R. C. (1980) Adv. Microcirc. 9, 45-75)]. In the last 5 years, a number of studies have provided evidence supporting this hypothesis. First, a series of elegant grafting experiments using chicks and quails demonstrated that the splanchnopleural mesoderm is able to generate hematopoietic cells and endothelium, while the paraxial mesoderm lacks this hematogenic capacity (Pardanaud, L. et al. (1996) Development 122, 1363-1371). Hematogenic activity in the former region is regulated by endoderm-derived cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor β1 (TGFβ1), whereas ectodermal factors such as epidermal growth factor (EGF) suppress it in the latter region (Pardanaud, L. and Dieterlen-Lievre, F. (1999) Development 126, 617-627). In the splanchnopleural mesoderm, cells expressing VEGF receptor 2 (VEGF-R2) were shown to form both hematopoietic and endothelial colonies (Eichmann, A. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 5141-5146). Furthermore, endothelial cells in the dorsal aorta generated CD45+ hematopoietic cells in vivo as evidenced by cell labeling experiments using DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) (Jaffredo, T. et al. (1998) Development 125, 4575-4583).
While similar in vivo grafting experiments are not possible in mammalian systems, it was found that hematopoietic cells were clustered at the ventral wall of the dorsal aorta in a 5 week-old human embryo (Tavian, M. et al. (1996) Blood 87, 67-72). More recently, Tavian et al. showed that CD34+ cells in the dorsal aorta and vitelline artery of human embryos are capable of generating hematopoietic cells in vitro (Tavian, M. et al. (1999) Development 126, 793-803). In mice, Nishikawa et al. recently showed that cells expressing Flk1 (mouse counterpart of VEGF-R2) and vascular endothelial cadherin (VECadherin) in the yolk sac and the P-Sp region of mouse embryos at 9.5 dpc gave rise to lymphohematopoietic cells in vitro (Nishikawa, S. et al. (1998) Immunity 8, 761-769). Similarly, Flk1+ hematogenic endothelial cells were generated from ES cells in vitro (Choi, K. et al. (1998) Development 125, 725-732; Nishikawa, S. I. et al. (1998) Development 125, 1747-1757). The idea that putative hemangioblasts express Flk1 arose originally from the finding that knockout mice lacking Flk1 exhibited severe defects in both hematopoiesis and vasculogenesis in the yolk sac (Shalaby, F. et al. (1995) Nature 376, 62-66). Furthermore, Flk1-null cells did not contribute to definitive hematopoiesis (Shalaby, F. et al. (1997) Cell 89, 981-990), although a recent report suggested that a significant number of hematopoietic cells can be induced from Flk1-null ES cells in vitro (Schuh, A. C. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 2159-2164).
In conformity with the critical role of TGFβ1 in the induction of hematopoiesis and vasculogenesis in chick embryos, knockout mice deficient in TGFβ1 also exhibited severe defects in both systems (Dickson, M. C. et al. (1995) Development 121, 1845-1854). Furthermore, gene disruption of the SCL/Tal-1 transcription factor caused defects in both hematopoiesis and vasculogenesis which were similar to those of Flk1 knockout mice (Porcher, C. et al. (1996) Cell 86, 47-57: Visvader, J. E. et al. (1998) Genes Dev. 12, 473-479). Mutant zebrafish devoid of the SCL/Tal-1 gene also showed similar defects (Liao, E. C. et al. (1998) Genes Dev. 12, 621-626) and forced expression of SCL/Tal-1 in zebrafish embryos resulted in the overproduction of hematopoietic and vascular cells (Gering, M. et al. (1998) EMBO J. 17, 4029-4045). Taken together, these studies clearly demonstrate the presence of hemangioblasts, the common precursors of both hematopoietic and endothelial cells, in fish, avian, and mammalian embryos and that VEGF-R2/Flk1, TGFβ1, and SCL/Tal-1 are essential for the development of hemangioblasts. However, the nature of hemangioblasts remains unexplored, in particular, there is yet no absolute evidence that LTR-HSCs are derived from hemangioblasts.